This project focuses on the role of DNA damage and repair in neurodegenerative conditions, in the pathological effects of alcohol abuse, and the in aging process. Studies in the past year continued have made progress in understanding the biological significance of a novel class of oxidative DNA lesions called cyclopurines (cPu) that are formed in DNA as a result of the hydroxyl radical. Unlike most oxidative DNA lesions, which are repaired by the base excision repair (BER) pathway, cPu are specifically repair by the nucleotide excision repair (NER) pathway. Thus these lesions are prime candidates for endogenous DNA lesions that cause the massive neurodegeneration observed in patients with the genetic disease xeroderma pigmentosum (XP) that lack the capacity to carry out NER. In the past year, we have defined another biological effect of cPu lesions that may be relevant to understanding the neurodegeneration in XP patients as well as the aging process. A single cPu lesion within the TATA box of a mammalian gene promoter was found to prevent binding by the TATA binding protein TBP, and also to strongly reduce gene expression in mammalian cells. This approach used in this work represents a paradigm for studying the biological effects of DNA lesions in functional important nontranscribed (FINT) DNA sequences on gene expression in mammalian cells. Ongoing studies are focusing on effects of these and other oxidative DNA lesions in other transcription factor binding sites such as CRE and the heat-shock elements. Biological Effects of DNA Lesions on Transcription in Mammalian Cells Given that there are thousands of DNA lesions present in cells under normal conditions as a result of normal metabolism and the inherent chemical instability of the DNA molecule, RNA polymerases must come into contact with DNA lesions on the template strand with some frequency. Exactly what happens when an RNA polymerase comes in contact with a DNA lesion in mammalian cells is not clear at present. To address this problem, we have developed a method to analyze the 3? end of nascent RNA transcripts associated with RNA polymerase II complexes in living mammalian cells. Using this method, we have obtained evidence stalling of an RNA pol II complex at some DNA lesions (e.g a thymine dimer) results in a transcript cleavage reaction, apparently mediated by the elongation factor SII. At a cyclopurine lesion, however, no evidence for SII mediated processing reactions was obtained. These data strongly support data using in vitro systems, and further indicate that the molecular mechanisms to deal with RNA pol II complexes stalled at DNA lesions may differ depending upon the specific lesion. A manuscript describing this work is in preparation. Double-Strand Break Repair and Neurodegeneration A new line of investigation that began this year focuses on the mechanisms of double-strand break (DSB) repair and its implications for neuronal death. Patients with the genetic disease ataxia telangectasia (AT) develop a progressive ataxia during childhood that results from severe loss of Purkinje neurons in the cerebellum. There is also evidence of abnormal develop of Purkinje neurons of AT patients. The mutated gene in AT patients, called ATM, encodes a protein kinase. Among the several targets of ATM kinase is a protein called Mre11 that is involved in DSB repair. Interestingly, patients with mutations in Mre11 also develop progressive cerebellar neurodegeneration. Studies begun in the past year have localized ATM, Mre11, and related proteins to the nucleus of Purkinje neurons in the human cerebellum. These finding have important inplications regarding the mechanims of neurodegeneration in AT and ATLD patients. A manuscript describing this work is nearing completion. Understanding the role of the ATM pathway in Purkinje neurons may also have implications for understanding the effects of alcohol in the developing brain. A gene-chip analysis of mRNA expression in an animal model of fetal alcohol syndrome (carried out by Susan Maier and Jim West at Texas A & M University) found that the most highly induced mRNA in the cerebellum of the alcohol treated offspring was Rad9. Rad9 has been shown to be a substrate for phosphorylation by ATM in response to DNA damage. These data suggest that the ATM pathway may play a role in responding to alcohol effects in the brain. Studies to investigate this possibility have been initiated. Genome Stability in the Brain An additional research focus begun last year concerns the stability the sequence of genomic DNA over time in non-dividing cells. It might be expected that in non-dividing cells the DNA sequence would be stable. However, the continued expression of the mismatch repair system in the adult brain we discovered previously (Marietta et al, Mol Brain Res ) suggests the potential for changes over time. To test this more directly, we have obtained a transgenic mouse from Dr. James Springer, (Univ. of Cincinnati), that has contains within its genome a constitutively expressed human placental alkaline gene containing a +1 frameshift mutation within a string of G residues. Reversion of this allele by loss of a single G makes a function protein which cane be detected in tissue sections using a histochemical assay. Using these mice, we have obtained clear evidence for frameshift mutations occurring solitary neurons as well, indicating that a somatic mutation occurred in the genome of these cells. Crossing these mice with knockouts lacking the mismatch repair protein PMS2 leads to a dramatic increase in cells with frameshift mutations, as would be expected based on the role of MMR in repairing frameshift mutations. Ongoing studies are looking at these animals over time to see if the numbers of mutant cells increase during aging. In addition, these animals will be exposed to various challenges including alcohol intoxication and withdrawal to see in these manipulations can change the sequence of genomic DNA in neurons. Finally, studies begun this year have identified several esophageal tumor cell lines whose growth is significantly slowed by ethanol. Ongoing studies are focused on assessing the capacity of these, as well as other transformed lines, to metabolize alcohol, with the specific goal of finding lines that express high levels of ADH4, the esophageal alcohol dehydrogenase. Once identified, these cell lines will be utilized in genetic toxicology studies to determine the relationship between alcohol metabolism and DNA damage, with the goal of understanding the relationship between alcohol consumtpion and esophageal cancer in humans.